Protein assay using microwave energy

ABSTRACT

A modification of the standard BCA protocol is provided that allows sensitive and reliable protein determinations in a matter of seconds utilizing a microwave oven to irradiate the samples. Methods of determining protein concentrations in a sample are disclosed comprising the steps of combining the sample with a BCA assay reagent in a sample container, placing the sample container into a microwave oven, irradiating the sample and measuring an absorbance value at 562 nm for the sample. Protein concentrations are then determined by comparing the absorbance value with a known value based on a calibration curve. In preferred embodiments, the calibration curve is generated by placing one or more standards in the microwave oven and irradiating the standards. Preferably, the step of irradiating the sample is carried out for a duration of less than about 60 seconds and most preferably, for a duration of about 20 seconds. The step of irradiating the sample most preferably comprises exposing the sample to microwave energy at a power level of between about 600 to about 650 watts at a frequency of about 2.45 GHz. In addition to the preferred embodiments using BCA assay reagents, it has been found that the incubation times of other protein assays, such as those based on the reaction with alkaline copper tartrate can also be significantly reduced by exposing the sample and the assay reagents to microwave irradiation.

The present invention relates to assays, and, more particularly relatesto protein assays exhibiting shortened reaction times.

Portions of the research related to the present invention may have beensupported by the United States through NIH Grant HD 15822. Accordingly,the United States may have certain limited rights to this invention.

This is a continuation of application Ser. No. 861,616, filed Apr. 1,1992, now abandoned.

BACKGROUND OF THE INVENTION

Assays are known that take advantage of a sensitive and highly specificinteraction between cuprous (Cu⁺¹) ions and the sodium salt ofbicinchoninic acid (BCA) for the determination of protein concentrationsin solution. See Smith, et al., "Measurement of Protein UsingBicinchoninic Acid", Analyt. Biochem. 150:76-85 (1985). At alkaline pH,proteins will reduce Cu⁺² to Cu⁻¹ in the presence of BCA, and thisreaction forms product that absorbs light strongly at a wavelength of562 nm. Since the production of Cu⁺¹ in the BCA assay is a function ofprotein concentration and incubation time, the protein content ofunknown samples may be determined spectrophotometrically by comparingthe sample absorption spectrum to that of known protein standards.

The ease with which an accurate BCA assay may be performed has made itpreferable to and of greater general utility than the Biuret, Lowry orBradford techniques reported in the prior art. See Lowry, et al."Protein Measurement with the Folin Phenol Reagent",J. Biol. Chem.193:265-275 (1951); "DC Protein Instruction Manual" Bio-RadLaboratories, Richmond, Calif. (modified Lowry Assay); and Bradford, "ARapid and Sensitive Method for the Quantitation of Microgram Quantitiesof Protein utilizing the Principle of Protein-Dye Binding", Analyt.Biochem. 72:248-254 (1976). This is especially true because of the lowlevels of interference caused by reagents commonly used in proteinpreparations, such as detergents, certain buffers and salts, and somereducing agents. See Pierce, "BCA* Protein Assay Reagent Protocol andInformation Manual 23220/23225" Pierce Chemical Co., Rockford, Ill.(1989); Smith, et al. "Measurement of Protein Using Bicinchoninic Acid",Analyt. Biochem. 150:76-85 (1985).

A particular concern in the use of protein assays involves instanceswhere such interfering compounds accumulate or partition differentiallyamong samples. For example, some detergents may associate preferentiallywith membrane proteins compared to cytosolic proteins duringchromatographic purification. In such situations, artificially highprotein levels are determined for membrane proteins, even if appropriatecorrections are made to account for the concentration of detergent inthe buffer. The use of the BCA assay obviates this problem since manydetergents do not interfere with the assay. See Smith, et al.,referenced above.

Kits taking advantage of the above-described BCA reaction are available,for example, from Pierce Chemical Co. (Rockford, Ill.). Three standardvariations of the BCA based protein assay are suggested by Pierce withincubation at either room temperature, 37 C., or 60 C. for a period of30 to 120 minutes. See Pierce, "BCA* Protein Assay Reagent Protocol andInformation Manual 23220/23225" Pierce Chemical Co., Rockford, Ill.,(1989). In general, it is known that the sensitivity of standard BCAassays increases with elevated incubation temperature and/or longerincubation time, and the assay can be easily adjusted to the range ofinterest. However, the time required for BCA protein analysis diminishesits utility for a number of reasons. For example, a large number ofsamples cannot be examined in a reasonable amount of time, nor cansamples be assayed from a system undergoing a changes on the order ofminutes, since the assay results would always lag the changes in thesystem. Additionally, the presently available assays are disruptive inprocedures that require protein determination at multiple intermediatesteps. It would therefore be desirable to reduce the incubation time ofprotein assays without diminishing the sensitivity or accuracy of theassay. It is therefore an object of the present invention to providemethods and apparatus for determining protein concentration based onknown assays that may be performed using incubation times on the orderof seconds, while retaining appropriate levels of sensitivity,reliability, accuracy and resistance to interfering compounds. It is afurther object of the present invention to provide assays that are easyto use and easily integrated into current laboratory practice.

SUMMARY OF THE INVENTION

In order to meet these and other objects, the present invention providesa modification of the standard BCA protocol that allows sensitive andreliable protein determinations in a matter of seconds utilizing astandard microwave oven to irradiate the samples. The methods andapparatus of the present invention are useful with other reactions, butfor the reasons discussed above, the improved BCA protocol represents apreferred embodiment for the determination of protein.

The present invention thus provides methods of determining proteinconcentrations in a sample comprising the steps of combining the samplewith a BCA assay reagent in a sample container, placing the samplecontainer into a microwave oven, irradiating the sample and measuring anabsorbance value at 562 nm for the sample. Protein concentrations arethen determined by comparing the absorbance value with a known valuebased on a calibration curve. In preferred embodiments, the calibrationcurve is generated by placing one or more standards in the microwaveoven and irradiating the standards. Preferably, the step of irradiatingthe sample is carried out for a duration of less than about 60 secondsand most preferably, for a duration of about 20 seconds. The step ofirradiating the sample most preferably comprises exposing the sample tomicrowave energy at a power level of between about 600 to about 650watts at a frequency of about 2.45 GHz. In general, the methods of thepresent invention may be adapted to analyze a plurality of samples. Inaddition to the preferred embodiments using BCA assay reagents, it hasbeen found that the incubation times of other protein assays can also besignificantly reduced by exposing the sample and the assay reagents tomicrowave irradiation. Thus, the present invention provides a generalmethod of determining protein concentrations in a sample based upon thereaction of protein with an alkaline copper tartrate solution and Folinreagent. These methods comprise the steps of combining the sample withassay reagent in a sample container, placing the sample container into amicrowave oven, irradiating the sample, and determining proteinconcentration, preferably by colorimetric analysis.

The present invention also discloses apparatus for conducting aBCA-based assay, comprising a microwave chamber for producing anhomogeneous microwave field, a photodetector chamber for the detectionof reaction product, and a controller. Most preferably, the apparatusalso includes either a means for regulating the duration time ofmicrowave exposure or a means for measuring the total amount ofmicrowave exposure, or both. In a preferred embodiment, a microwavedetector is provided for measuring the duration time of microwaveexposure and providing a signal indicative of an intensity value ofmicrowave energy in the microwave chamber. In certain automatedembodiments, a flow system is connected to the controller and thecontroller controls the flow of one or more reagents from the flowsystem into a sample container.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a typical plot of absorbance versus bovine serumalbumin concentration obtained using the BCA assay of the presentinvention.

FIG. 2 illustrates the effect of increasing microwave irradiation timeon the BCA assay of the present invention. Open triangles=1.0 mg/ml;open circles=0.8 mg/ml; and closed circles=0.2 mg/ml;

FIG. 3 is a bar graph illustrating the effect of common reagents on theBCA assay of the present invention.

FIG. 4 is a schematic of a preferred embodiment of the apparatus of thepresent invention.

FIG. 5 is a plot similar to FIG. 1 showing the results of a microwaveenhanced Lowry assay compared to a standard Lowry assay. Opensquares=room temperature (RT) mean; closed squares=microwave (μw) mean.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As noted above, three standard variations of the BCA-based protein assayuse incubation at either room temperature, 37 C., or 60 C. for a periodof 30 to 120 minutes. Since sensitivity is proportional to incubationtemperature and time, the standard assay can be adjusted to the range ofinterest. The present invention provides a refinement to this protocolby taking advantage of the accelerated reaction rate that it has nowbeen found is produced by microwave irradiation. Preferably, theirradiation is carried out using a household microwave oven, such as theWhirlpool Model RJM7450 which has a capacity of 1.3 cubic feet andproduces 650 watts of microwave energy at a wavelength of 2.45 GHz.However, other ovens or microwave heating chambers having differentcapacities, power levels and wavelengths may also be used with thepresent invention. For example, a 0.8 cubic foot General Electricmicrowave oven producing 600 watts (Model JEM18F001) was used to obtainthe data shown in FIG. 1. As illustrated by the following Example andexplained in further detail below, irradiating the samples in ahousehold microwave oven allows the accurate, BCA-based determination ofprotein concentrations in a matter of seconds.

EXAMPLE I

A typical standard curve generated with the present invention usingbovine serum albumin (BSA) dissolved in water is shown in FIG. 1. BSA isavailable, for example, from Sigma Chemical Co., St. Louis, Mo. Samplesof BSA, in a volume of 100 μl, were prepared in triplicate and combinedwith 2 ml of BCA reagent in polystyrene Rohren tubes such as thoseavailable from Sarstedt Inc., Newtown, N.C. The tubes were placed in aplastic holding rack. It should be noted that metal holding racks shouldnot be used in a an environment where they will be exposed to microwaveradiation. A beaker containing 100 ml of room temperature water was alsoplaced in the oven as an energy buffer to absorb some of the excessmicrowave energy. The provision of an energy buffer keeps thetemperature of the samples from rising too rapidly and helps to maintainuniform exposure of the samples to microwave radiation. This techniqueis a known expedient in microwave heating techniques that havepreviously been applied as time-saving and efficiency-enhancingprocedures. See, e.g., Boon, et al. "Microwave Cookbook: The Art ofMicroscopic Visualization" Coulomb Press Leyden, Leiden, Netherlands(1989). It appears, however, that while placing the test tube rackitself into a tray of water to better homogenize the water load andavoid any "hot spots" in the samples could be useful in some specificapplications, in general, this step has been found to be unnecessary.The samples were placed in the center of the microwave oven andirradiated for 20 seconds on the highest setting. Absorbance values at562 nm were measured within 5 minutes after irradiation and plottedagainst BSA concentration.

FIG. 1 shows a linear standard curve that would be suitable for thedetermination of protein concentration in unknown samples preparedduring the same microwave run. Some variability in absorbance valuesobtained for a given BSA concentration was found under certainconditions, e.g., using different microwave ovens, changing the batch ofBCA reagents, slightly lengthening or shortening irradiation times, orchanging the size or starting temperature of the water load. Thisvariability is easily compensated for by generating a BSA-based standardcurve with each microwave determination as an internal control.

Using the microwave BCA procedure of the present invention, the effectof varying irradiation time on the reaction can also be determined.Referring now to FIG. 2, three concentrations of BSA (in water) weretested, and each time point was determined from a single irradiationtrial, with the water load replaced before subsequent determinations. Asingle batch of BCA reagent was used throughout the experiment. Eachvalue is the mean of triplicate samples ± standard deviation. Thus, FIG.2 shows the rate of reaction for three different concentrations of BSAover 5-30 seconds of microwave exposure. It can be seen that absorbancevalues increased for each BSA concentration as a second order functionof irradiation time. Curve fitting was done using Cricket Graph software(Cricket Software, Philadelphia, PA). The second order equations yieldeda correlation coefficient =0.99 for each BSA concentration. Since astandard curve may be generated from BSA dilutions that are irradiatedfor specific times, in practice, the duration of microwave exposure canbe selected to correspond to the sensitivity range desired, with longertimes being more suitable for lower protein concentrations. The durationof 20 seconds chosen for the preferred embodiment of the microwaveprocedure described in Example I and for collecting other data discussedherein is more sensitive than a standard assay such as the Pierce assaydiscussed above, which is performed at room temperature.

Therefore, when further increases in sensitivity are desired, microwaveexposure times are increased. As noted above, the duration of theirradiation is determined by using BSA test solutions in the range ofprotein concentrations expected until desirable absorbance values areobtained. By increasing microwave exposure time, it is thus possible tosubstantially increase assay sensitivity while still keeping the assaytime below 60 seconds. The surprising ease with which sensitivity may beadjusted within extremely short time frames makes the microwave BCA thequickest, most flexible assay available for protein determinations.

As noted above, BCA assays generally do not suffer from inaccuraciescaused by common laboratory reagents interfering with the assay. It hasbeen reported in the literature, however, that certain compoundscompromise the accuracy of BCA based assays. See Smith et al.,"Measurement of Protein Using Bicinchoninic Acid," Analyt. Biochem.150:76-85. (1985). Similar compromises are also likely to be true forthe microwave BCA assay disclosed by the present invention. Moreover,because of the procedural variation, some originally non-interferingcompounds could interfere with the microwave assay. For example,materials that absorb microwaves very efficiently might be particularlytroubling since they would tend to shield samples from uniform exposure.In addition, compounds which are degraded or converted during themicrowave procedure, as well as any that have altered interactions withother assay components, might also substantially affect the assayresults. The interference of such materials can be predicted byconsidering the relative permittivities and dielectric characteristicsof the samples being assayed. The determination or approximation ofthese microwave absorption characteristics are well known. However,interference, as with treatment duration, is most easily assessedempirically by directly determining the effect of a given additive onthe accuracy and sensitivity of the assay.

The extent of the interference caused by selected compounds is shown inFIG. 3. These results are derived from a comparison of the absorbancevalues produced by 200 μg/ml BSA solutions containing various reagentsduring a 20 second microwave assay, as discussed above in Example I, tothat produced by BSA in water alone. Stock preparations of BSA wereprepared to give a final BSA concentration of 200 μg/ml in the presenceof the reagents. The data presented are mean ± standard deviation (n=7).The treated samples were compared to a control (i.e., BSA in wateronly), and statistically significant differences are indicated withasterisks. By two-tailed Student's t-tests, SDS (1.0%), Triton X-100(1.0%), Hepes buffer (0.1 M, pH 7.2), and dithiothreitol (DTT, 1 mM) didnot interfere with the assay. It has been found, however, that Tris-HClbuffer (0.1 M, pH 8.0 or 11.0), glycine (1 M, pH 11.0), andβ-mercaptoethanol (βME, 1.0%) all substantially affected the reaction.It has further been found, however, that for solution containingTris-HCl or glycine, linear graphs of BSA concentration versusabsorbance are obtained by including these compounds in the standard andall samples. On the other hand, βME interfered to such a large extentthat the assay was not usable in its presence. As will be readilyunderstood by those of ordinary skill, any compound added to themicrowave BCA assay disclosed herein is easily checked for interferencein a similar manner to assess its effects on both sensitivity andaccuracy.

Thus, the present invention provides a microwave BCA protein assayprotocol that most preferably comprises the steps of 1) combiningsamples and BSA standards with BCA assay reagent in polystyrene tubes2); placing samples into a nonmetallic rack in the center of a microwaveoven, along with a tray or beaker containing about 100 ml of roomtemperature water; 3) irradiating the samples, most preferably for about20 seconds on the highest microwave setting; 4) measuring the absorbanceat 562 nm for each sample; and 5) determining protein concentrationsbased on a BSA calibration curve. The microwave protein assay disclosedby the present invention is suitable for all situations where a BCAassay is presently used. The incubation time for a BCA-based proteinassay using the methods known in the prior art is about 30-60 minutes,whereas a microwave BCA-based protein assay performed in accordance withthe present invention requires incubation of less than 60 seconds. Theability to determine accurate protein concentrations in a relativelyshort time greatly facilitates routine assays and improves efficiencywhen protocols require protein determination at multiple intermediatesteps.

The microwave assay discussed above in Example I has been used togenerate chromatograms during protein purification and for generalprotein determinations, e.g., before electrophoretic analysis. The assaydisclosed by the present invention consistently yields reliable resultsthat are comparable to those obtained by the standard, 30 minute Pierceprotocol discussed above. Moreover, the present invention is very easilyadapted to any microwave oven and can be adjusted to cover a wide rangeof protein concentrations. Since the duration of the assay is so shortrelative to the incubation times found in the prior art, it is possibleto run assays using several irradiation times and water loads todetermine the specific conditions required by the particular microwaveoven and samples being assayed. The microwave BCA assay of the presentinvention should therefore prove to be extremely useful in laboratoriescurrently using BCA-based assays for protein determinations.

Another aspect of the present invention is the design of a microwaveprotein assay apparatus that advantageously uses the microwave enhancedassay of the present invention. The standard BCA assay disclosed in theSmith et al. article referenced above is a time-based assay whichresults in the continued production of product (purple color) over time.Therefore, as shown in FIG. 2, the duration of microwave exposure--whichaccelerates the rate of product formation--is directly related to thesensitivity of the assay. Thus, for a BCA-based assay conducted inaccordance with the present invention, an apparatus must contain a meansfor regulating the time of microwave exposure or a means for measuringthe total amount of microwave exposure, or both. In the absence of oneof these two means, a standard curve must be generated each time on ormore samples are assayed; with at least one of these means, it becomespossible to calibrate apparatus made in accordance with the presentinvention based on total microwave exposure (duration and fieldstrength) so that standards need only be run periodically.

Referring now to FIG. 4, a schematic of an apparatus made in accordancewith the present invention is shown. The apparatus preferably comprisesa microwave chamber 10 that preferably produces an homogeneous microwavefield, a photometer chamber 20 such as a spectrophotometer, colorimeteror fluorimeter for the detection of reaction product, and a controller30. The specific dimensions and specifications of each of thesecomponents would be readily chosen by one of ordinary skill and arelargely dependent on the maximum number of samples to be assayed. Itshould be noted, however, that a device made in accordance with thepresent invention is not necessarily comprised of three physicallyseparate devices.

In a preferred embodiment, the microwave chamber 10 preferably operatesat about 2.45 GHz, which is the standard frequency of commerciallyavailable microwave ovens. The production of relatively uniformmicrowave fields is well known to those of ordinary skill, as is that ofthe construction and operation of microwave detectors for thisfrequency. It should be noted that 2.45 GHz has been approved by theU.S. Federal Communications Commission for general use. In householdmicrowave ovens, homogeneity of the electromagnetic field is obtained byscattering the microwave beam. Typically, microwave energy istransmitted via a waveguide to the top of the oven chamber and a fanwith angled, reflective blades is spun in the path of the beam exitingfrom the waveguide, causing the energy to scatter. A relativelyhomogeneous field in three dimensions is completed by microwavesbouncing off the walls of the chamber in all directions. An alternativemethod for the production of an homogeneous microwave field utilizesgeometrical optics and the placement of reflective surfaces in the lightpath. This method is used in many light sources for optical devices, butis not used in microwave devices due to alignment difficulties, spatialconsiderations and expense. Those of ordinary skill will appreciate thatthere are many variants to these methods of producing an homogeneousmicrowave field which may be applied in the design of apparatus made inaccordance with the present invention. The principal concern, however,is that the field be substantially homogenous and that the chamber bedesigned so that all of the samples "see" the same microwave field.

Preferably, a microwave detector 12 should be included in the chamber10. The detector 12 provides feedback of the intensity of microwaveenergy, which may vary over the lifetime of the microwave source, or dueto power fluctuations or other causes. The detector 12 also providesinformation concerning the duration of microwave exposure, which isrelated to assay sensitivity in some cases.

In those embodiments wherein the photometer 20 is a spectrophotometer,the device is preferably comprised of a light source, a wavelengthfilter, and a detector connected to the controller 30 which convertstransmittance readings into values which correspond to the opticaldensity (OD) of the sample. The optical density value indicatesconcentration in each of the protein determination methods mentionedabove; therefore, a microwave protein assay apparatus would preferablycontain a spectrophotometric detector. The photometer 20 may beconstructed to detect emissions from multiple samples from an assay intwo different ways: in parallel, using an array of detectors to measuremany samples at once, or by moving a plurality of samples past a singledetector. The wavelength used for detection may be selected by theplacement of individual filters in the detector light path or by the useof monochromators. Commercially available multiwell microtiter platereaders (using filters) can measure and record the optical density ofsamples at a rate of approximately 50 samples per minute using a singledetector. These readers are generally sold as microplate readers bycompanies such as Artek Systems Corp., Farmingdale, N.Y. and Bio-RadLaboratories, Richmond, Calif. However, those of ordinary skill willunderstand that this rate of sample throughput is not limited and it maybe possible to increase this rate further. The assay time for a singlesample using either parallel or single detector configurations isestimated to be less than 30 seconds from the time the sample is placedin the device to the time a result is available. For large scale use,the number of assays that may be performed per minute is a function ofthe capacity of the microwave chamber 10 and the detector 12. Since, intheory, the microwave chamber 10 may be designed to hold as many samplesas desired, the rate limiting step is detection, and as noted, rates fora single detector machine may be on the order of 50 samples per min. Fora parallel machine, this rate may be multiplied by the number ofdetectors used. Thus, it can be seen that the present invention permitslarge numbers of samples to be performed in a matter of minutes if aplurality of detectors 12 are used in parallel.

The design of an apparatus for performing assays in accordance with thepresent invention may involve a separate microwave chamber 10 and adetection chamber containing the photometer 20, with a mechanical meansto move samples from one compartment to the other, or it may involve acombined chamber where both irradiation and detection occur.

FIG. 4 also illustrates a computer-operated controller 30 for themonitoring of calibration and assay status, as well as the storage ofspecific assay parameters, results and, if desired, the control ofreagent flow into each sample using an automated flow system 40 thatpreferably comprises a controlled valve 42 connected to the controller30. Those of ordinary skill will be familiar with the construction andoperation of automated flow systems and will further understand that theoutput of the flow system 40 illustrated may be directed to the samplesafter they are placed in the chamber, or, alternatively, may be directedto the samples when they are in another location, prior to their beingtransferred into the microwave chamber 10.

Thus, for example, in a preferred embodiment, a technician places avolume of sample into each well of a microtiter plate and places thisplate into the microwave chamber 10. The addition of reagents would becontrolled through valve 42 built into the apparatus so that each samplereceived the correct volume of reagent from a reservoir. The controller30 would then turn on the homogeneous microwave field, monitor itsintensity and duration using the detector 12 and turn off the fieldafter a predetermined exposure. The controller 30 then activates thephotometer 20 and reads the optical density of each sample, stores thisvalue, converts the raw data into predetermined units, and displays dataindicative of the results. Upon completion of an analysis cycle, thecontroller 30 preferably flushes the flow system 40 and, upon initiationof a new analysis cycle, the controller 30 would prime the flow system40 and calibrate the apparatus for a range of protein concentrations anda given batch of reagents.

An additional component of the controller 30 is a hard copy outputdevice 32 for the generation of reports concerning the status of theapparatus, results from particular assays and the like.

The surprising results obtained using the present invention do notappear to correlate to other prior art assay techniques. As previouslynoted, the present invention permits sensitivity to be readily adjustedby varying the time of the microwave irradiation. As shown in FIG. 5, acomparison has been made of the results obtained using a prior art Lowryassay, described above, performed at room temperature with an incubationtime of 15 minutes with a Lowry assay performed by exposing the sampleand reagents to microwave energy in a manner similar to that describedabove in Example I, using irradiation time of 10 seconds. The Lowryassay is apparently an end point assay which results in a maximal levelof product formed over time; therefore, as seen in FIG. 5, the durationof microwave exposure only accelerates the reaction as it progresses tocompletion and does not affect assay sensitivity. Thus, the measurementof protein concentrations using a microwave Lowry-based reaction may notrequire a built in calibration system. A Lowry-based apparatus would becalibrated periodically by running known standard proteins. The standard(non-microwave) incubation time for a Lowry based protein assay is 15-30minutes; a microwave-Lowry based protein assay requires incubation of 10seconds. Thus, it has been discovered that the incubation time may besignificantly reduced. It should be noted, however, that extendedirradiation times of more than 30 seconds of Lowry reaction mixturescontaining BSA results in excessive heat production and the formation ofa white precipitate of uncharacterized composition.

Thus, the present inventions also discloses methods for determiningprotein concentrations samples using a Lowry assay or other assays basedupon the reaction of protein with an alkaline copper tartrate solutionand Folin reagent. These embodiments of the present invention comprisesteps of combining the sample with the appropriate assay reagents in asample container, placing the sample container into a microwave oven andirradiating the sample. Protein concentration is then determined,preferably by colorimetric analysis, as is well known to those ofordinary skill.

Both the BCA and Lowry assays are extensions of the Biuret reactiondescribed by Gornall et al. in the J.Biol.Chem. 177:751 (1949). The BCAand Lowry assays allow for higher sensitivity over the Biuret reaction.The reason for the accelerated reactions upon exposure to microwaveradiation is likely due to an increased rate of production of Cu¹⁺ ;therefore, a microwave apparatus like those described above may beuseful in standard Biuret reactions for the reduction of incubationtimes. The standard (non-microwave) incubation time for a Biuretreaction based protein assay is 30 min; a microwave-Biuret reactionbased protein assay would therefore theoretically require a shorterincubation time.

In conclusion, a microwave-based protein detection system may bedesigned to run any or all of the available protein determinationreactions, most preferably a BCA assay but also either a Lowry assay orother assays based upon the Biuret reaction. The apparatus of thepresent invention may be designed to accommodate a large number ofsamples with high speed and represents an enhancement over any availablesystem by virtue of the reduced incubation times necessary. Theapparatus of the present invention may be designed to automaticallycarry out may of the steps necessary for the estimation of protein. Thisapparatus may be generally useful in assays where incubation of amixture results in the production of a product with known transmittanceproperties (e.g. colorimetric enzyme assays, detection of sugars orother chemical compounds, etc.); adaptation of this apparatus to otherassays may be done by simply controlling microwave exposure and thewavelength of detection.

Although certain embodiments of the present invention have been setforth with particularity, these examples are for the purpose ofillustrating the invention and are not meant to be limiting.Accordingly, reference should be made to the appended claims in order todetermine the scope of the present invention.

What is claimed is:
 1. A method of determining protein concentrations ina sample comprising the steps of:combining the sample with a BCA assayreagent in a sample container; placing the sample container containingthe sample and reagents into a microwave oven; irradiating the samplefor about 20 seconds in the microwave oven, whereby a reaction betweenthe sample and the reagent produces a product; measuring an absorbancevalue for the sample at a predetermined wavelength; and determiningprotein concentrations by comparing the absorbance value with acalibration curve representing absorbance as a function ofconcentration, created for the predetermined wavelength.
 2. The methodof claim 1, wherein the calibration curve is generated by placing one ormore standards in the microwave oven and irradiating the standards. 3.The method of claim 1, wherein the step of placing the sample containerinto the microwave oven further comprises placing a quantity of water inthe microwave oven.
 4. The method of claim 1, wherein the predeterminedwavelength is 562 nm.
 5. The method of claim 1, wherein the step ofirradiating the sample comprises exposing the sample to microwave energyat a power level of between about 600 to about 650 watts.
 6. The methodof claim 1 wherein the step of irradiating the sample comprises exposingthe sample to microwave energy at a frequency of about 2.45 GHz.
 7. Themethod of claim 1, wherein protein concentration is determined in aplurality of samples by combining the plurality of samples with a BCAassay reagent in a plurality of sample containers, placing the samplecontainers into the microwave oven, simultaneously irradiating theplurality of samples, measuring an absorbance value at 562 nm for eachsample, and determining protein concentration in each sample bycomparing the absorbance value with a calibration curve representingabsorbance as a function of concentration.
 8. A method for determiningprotein concentrations in a plurality of samples using apparatus forconducting a BCA-based assay that includes a microwave chamber, themethod comprising the steps of:calibrating the apparatus for a range ofprotein concentrations and a given batch of reagents; placing a volumeof a sample into each well of a multiwell plate to create the pluralityof samples, wherein each well has a capacity sufficient to retain thevolume of the sample and a predetermined volume of the reagents; placingthe multiwell plate into the microwave chamber; adding the predeterminedvolumes of the reagents to each of the plurality of samples through aflow system comprising controlled valves; activating a homogeneousmicrowave field within the microwave chamber; monitoring an intensityand duration of the microwave field; deactivating the microwave fieldafter a predetermined exposure time period of about 20 secondsactivating a detection system to determine an absorbance value for eachone of the plurality of samples; storing the absorbance value andconverting the absorbance value to predetermined units indicative of theprotein concentration in each sample; and displaying data indicative ofthe predetermined units.
 9. The method of claim 8, further comprisingthe steps of:flushing at the flow system; priming the flow system; andrecalibrating the apparatus for a range of protein concentrations and agiven batch of reagents.
 10. A method of determining proteinconcentrations in a sample based upon a reaction of protein with areagent that initiates a Biuret reaction comprising the stepsof:combining the sample with assay reagent in a sample container;placing the sample container into a microwave oven; irradiating thesample for about 20 seconds; and determining protein concentration. 11.The method of claim 10 wherein the reagent comprises at least analkaline copper tartrate solution and a Folin solution.
 12. The methodof claim 10 wherein the step of determining protein concentrationcomprises colorimetric analysis.